Stable insulin material

ABSTRACT

A substantially dry and substantially defatted solid pancreatic tissue material which contains active insulin in hormonal levels corresponding to the amount in an equivalent amount of fresh pancreas, but said solid material being substantially free of pancreatic enzymes. The dry product is stable at room temperature and therefore provides an improved insulin source material which can be more economically handled because of markedly smaller weight and easier handling properties, and which can be later processed to separate the insulin. The improved, stable insulin source material is prepared by deactivating the pancreatic enzymes with acid treatment, then removing substantially all the water from the pancreas by azeotropic distillation; and removing substantially all the fat by dissolving in a fat organic solvent which is also used to form the azeotrope. The insulin present in the dry, stable material may then be conventionally separated and converted into acceptable pharmaceutical forms.

United States Patent Levin et a].

[ 51 July 11, 1972 [54] STABLE INSULIN MATERIAL [72] Inventors: EzraLevin, Champaign, 111.; Ezra Levin,

Champaign, Ill.

[73] Assignee: Violin Corporation, Champaign, III.

[22] Filed: Oct. 9, 1967 [2]] Appl. No.: 673,958

52 misc: ..424/110,424/17s 511 men. ..A61k 17/04 [58] FieldofSearch..424/llO,l78;l67/75 [56] References Cited UNITED STATES PATENTS1,027,790 5/1912 Zuelzer ..260/l 12.5 1,520,673 l2/l924 Walden...424/1l0 2,503,312 4/l950 Worsham et al ..99/l57 OTHER PUBLICATIONSSizer, American Brewer, Vol. 85, No. 5, pp. 29- 33, 77, May, 1952.

Primary Examiner-Albert T. Meyers Assistant ExaminerDoris J. FunderburkAt!0rneyBurmeister & Kulie [5 7] ABSTRACT The improved, stable insulinsource material is prepared by deactivating the pancreatic enzymes withacid treatment, then removing substantially all the water from thepancreas by azeotropic distillation; and removing substantially all thefat by dissolving in a fat organic solvent which is also used to formthe azeotrope. The insulin present in the dry, stable material may thenbe conventionally separated and converted into acceptable pharmaceuticalforms.

8 Claims, No Drawings STABLE INSULIN MATERIAL This invention relates toan insulin source material and to a method for preparing the product. Inparticular, the invention relates to a dry and defatted source materialwhich has indefinite shelf life and from which the insulin may beextracted by conventional process steps.

The common practice now for separating insulin from pancreas is to startwith fresh pancreas which is usually frozen. While many variations areknown and practiced, such fresh pancreas is treated by a step which isgenerally characterized as an acidified alcohol extraction. The aciddeactivates the enzymes but not the hormone, insulin and the alcoholextracts the insulin. A conventional ethyl alcohol and hydrochloric acidextraction is described, for example, in US. Pat. No. 2,695,861. Thispatent also teaches a phosphoric acid modification step which allowssuch an acid to be later removed as an insoluble ammonium salt. Otherreview articles on this subject are found in Research Today, Vol. 10,No. 3,58-86 (1954); and Science, Vol. 129, l340-l344, 1959).

The use of fresh pancreas, of course, presents problems in handling andstorage. If the fresh pancreas is not immediately processed, then extraefforts must be made to preserve the pancreas as a starting material forinsulin extraction. The common procedure is to freeze the pancreas ifthe extraction process is not to be immediately conducted. In practice,the pancreas is commonly frozen before extraction in the commercialprocesS.

It is evident that handling fresh pancreas presents the problem ofmanipulating large masses because large amounts of pancreatic tissue arerequired in order to obtain a given amount of extracted insulin hormoneactivity. This bulk handling is a serious problem and its economicimplications include the cost of transportation and the man powerrequired in handling and treating such fresh frozen pancreatic tissues.This is in addition to the refrigeration facilities which are requiredto both quick freeze the pancreatic tissues and to maintain thepancreatic tissues in the frozenstate until it is used to. extract theinsulin.

An important contribution to this art would be to provide a sourcematerial for insulin extraction which is equivalent to fresh pancreas inthe levels of active insulin which can be extracted from a given amountof source material, but which is of much lesser weight and has improvedhandling properties.

It is accordingly one primary object of the invention to provide aninsulin source material which is pancreatic tissue, but which is a drysolid having a markedly reduced bulk and weight while still retaininginsulin activity of the same order as found in the corresponding amountof fresh pancreatic tissue.

A still further object of the invention is a method for preparingsubstantially dried and substantially defatted pancreatic tissue sourcematerial which is substantially free of enzymes but which containsinsulin; and from which the insulin may be extracted by conventionalprocess steps in amounts equivalent to those obtained from correspondingamounts of fresh pancreas.

The foregoing objects are realized, as well as still other objects whichwill occur to practitioners, by the invention which will now bedisclosed.

Insulin has been extracted from fresh or freshly frozen pancreas byconventional acid alcohol extraction, but it is now provided that freshanimal pancreas may be manipulated by novel process steps which includedefatting and desiccating the fresh tissue to obtain a dry comminutedfonn having unimpaired insulin level activities. It is known in the artto obtain defatted and desiccated animal tissues such as pancreas byremoving water as an azeotrope of water and an organic fat solventwhile, simultaneously, extracting the fat by the same solvent. Such aprocess has been disclosed in US. Pat. Nos. 2,503,313 and 2,619,425issued to the present applicant. The dried and defatted pancreatictissue material obtained by the teachings of such patents containsconcentrated levels of pancreatic enzymes such as protease. lipase,amylase, collagenase, elastuse and others. This dried product, however,contains substantially no insulin because the insulin is (leaclivatetlby the foregoing pancreatic enzymes.

A process has been devised according to the present invention wherein adry and defatted pancreatic tissue product is obtained which issubstantially free of such pancreatic enzymes but which, surprisingly,has high levels of insulin which can be successfully extracted evenafter extended storage of the tissue material at room temperature.

By the practice of the method, the pancreatic enzymes, are deactivatedin fresh pancreas by contacting fresh pancreatic tissue with sufficientamounts of acid to effect the deactivation. The acid treated tissue isthen defatted and dehydrated by removing the water as an azeotrope witha fat organic solvent as disclosed in the foregoing US. Pat. Nos.2,503,313 and 2,619,425. The removal of the water as an azeotrope isconducted at moderate temperatures so as not to deactivate the insulin,say below about F or below about 38 C.

While the step of deactivating the pancreatic enzymes with an acid maybe practiced in a variety of ways, it is presently preferred to combinethe pancreas with a strong mineral acid such as sulphuric, hydrochloric,nitric or the like. The acidcontacted pancreas is then broken into smallparticle sizes as by passing through a grinder. The comminutedpancreatic tissue is then delivered for immersion into a body of boilingfat organic solvent in a depressurized area so that the solvent boils ata reduced temperature.

An azeotrope of the fat organic solvent and the water is formed andremoved in a distillation zone adjoining the body of boiling fat organicsolvent. It is preferred to deliver a given amount of pancreatic tissuematerial to several times its volume of fat organic solvent sosufficient fat organic solvent is present to both form the azeotrope andto extract the fat from the pancreas. In general, three or four volumesof solvent are preferably present at any time for a given volume oftissue material, and means may be provided for replacing spent solventcontinuously or otherwise. The azeotropic distillation is continueduntil the water content of the pancreatic tissue is reduced to desirablelevels, generally below about 6 percent by weight. At this time the fatcontent of the pancreatic tissue has also been reduced to desirably lowlevels of about less than 2-3 percent by weight.

The substantially defatted and desiccated pancreatic tissue is separatedfrom the body of fat organic solvent by means such as draining thesolvent away from the depressurized areas. Additional volumes of solventmay then be introduced in a rinsing operation to remove any occluded fatwhich may be present. The solvents used for rinsing may also be removedby draining or the like, and the pancreatic material may then be airdried to obtain the comminuted solid. Such a solid may be stored with orwithout further comminution. In general, it is preferred to provide apowdered solid form which even further reduces the bulk for handling.Such a fine powdered form also facilitates subsequent extraction of theinsulin therefrom by conventional process steps.

It is generally preferred to prepare the pancreatic tissue product frompork or beef pancreas, and it is preferred to use halogenatedhydrocarbons of the type described in the foregoing US. patents. Suchsolvents include propylene dichloride, trichlorethylene,perchlorethylene and others. The preferred solvent is ethylenedichloride. Non-halogenated solvents may, however, also be used such asbenzene, hexane, pentane, toluene, cyclopentane, cyclohexane and stillothers. If desired, the dehydration and defatting process may beinterrupted to further reduce the particle size by mechanical means suchas a grinder or ball mill. Such further comminuted particles may then bereturned to the body of fat organic solvent which is heated in acontainer in the de-pressurized area, and defatting desiccation bydistillation may be concluded to the desired low levels.

The following examples are presented to illustrate some embodimentswhich may be followed in the practice of this invention. Suchembodiments should be construed as being only illustrative teachings,even though they are the best ones presently contemplated for servingtheir particular purposes.

EXAMPLE 1 Insulin Source Material To 500 gms of frozen pork pancreasglands is added 16.5 ml of 9N sulphuric acid, and the acidified mixtureis passed through a meat grinder to provide intimate mixing of the acidwith the pancreatic tissue. This acid treatment destroys the pancreaticenzymes but not the insulin.

Following such thorough mixing, the acidified pancreas is placed in aflask container to which a condenser and a source of vacuum areconnected. Ethylene dichloride is introduced into the flask in an amountof 2 /2 to 3 liters which amount will provide a body of boiling fatorganic solvents sufficient to attain desired agitation of the acidifiedpancreas in the solvent.

A sufficient vacuum is applied to maintain 23 inches of vacuum. Heat isapplied to the flask sufficient to boil the body of ethylene dichloride,and additional amounts of ethylene dichloride are added to the flask asthe azeotrope is formed and removed through the condenser. Thetemperature of the distillation is conducted below 100 F. The azeotropicremoval of water is continued until the resulting pancreatic tissuematerial, also called pancreatic meal, is substantially dry, that is,having a moisture content of no more than about 6 percent by weight.

The pancreatic meal is separated from the body of fat organic solvent byfiltering the mixture on a Buchner funnel. Any residual or occluded fatis removed from the pancreatic meal by a plurality of washing steps withethylene dichloride, and the meal is then air dried to provide a yieldof about 100 gms of substantially dried and defatted pancreatic tissuematerial in which the active insulin level is comparable to that whichwould be extracted from a corresponding amount of fresh pancreas.

EXAMPLE 2 lnsulin Extraction from Source Material A substantially driedand defatted source material, which is obtained by the process steps ofExample 1, is used as an intermediate or startinG material forconventional insulin extraction. A crude insulin concentrate is obtainedby depositing a sample of such material in several volumes of water toeffect an aqueous extraction. The extracted insulin is then precipitatedeither by adding an alcohol-ether mixture or by altering the pH to 5.2to effect isoelectric precipitation.

It will be noted from the foregoing Example 1 that the resulting driedand defatted source material has a weight about one-fifth of theoriginal fresh pancreas. This is a striking reduction in the weight andthe mass of the material which can be used for subsequent insulinextraction. This dimensional reduction, of course, leads to immediatesavings in transportation and handling, and eliminates the necessity offreezing and refrigeration. These foregoing improvements relative to theweight and bulk of the source material are accompanied by the very realimprovement that there is no substantial alteration in the hormonalcontent over what was present in the fresh pancreatic starting material.In other words, the insulin activity level is not substantiallydiminished in the improved, dried and defatted solid form. A new productand process are therefore provided for handling tissue material withoutsubstantially lowering the expected insulin content.

There are many immediately apparent advantages of the new product, andone which should be explicitly described is the feasibility ofextracting the insulin therefrom at a time and place removed from wherethe source material is prepared. The source material, for example, maybe shipped to a country where it can be extracted by conventionalprocess steps, and then converted to consumer acceptable forms. Suchconsumers are then relieved of the necessity of purchasing finalpharmaceutical forms from a supplier who first extracts the insulin fromfresh pancreas.

What is claimed is:

l. A stable and solid pancreatic product which provides an insulinsource for subsequent utilization including comminuted pancreatic tissuematerial, wherein said tissue material is substantially free of water,

substantially free of fat,

substantially free of pancreatic enzymes, and

characterized by an insulin content which is equivalent to the amount ofinsulin available in the corresponding amount of fresh pancreas, saidinsulin retaining its hormonal activity in a manner equivalent to thehormonal activity of insulin obtained from a corresponding amount offresh pancreas.

2. A product as in claim 1 in which the dry product has a water contentof less than 6 percent by weight, and has a fat content of less than 3percent by weight.

3. A product as in claim 2 in which the comminuted tissue material is inthe form of a powder.

4. A product as in claim 1 in which the pancreatic material lS selectedfrom the group consisting of beef and pork pancreas.

5. A method for preparing a stable insulin source material from whichactive insulin may be later separated without any substantial changes inthe insulin levels present in an original source of fresh pancreas,including the steps of contacting a body of fresh pancreas containinginsulin, pancreatic enzymes, water and fat with acid of a type and insufficient amount to deactivate substantially all the pancreaticenzymes,

combining the enzyme deactivated fresh pancreas in the comminuted statewith a body of boiling organic liquid fat solvent selected from thegroup consisting of ethylene dichloride, trichloroethylene, propylenedichloride, perchloroethylene, benzene, hexane, pentane, toluene,cyclopentane and cyclohexane in a distillation zone which isdepressurized so that the said organic liquid fat solvent boils below F,said organic liquid fat solvent forming an azeotrope with water,

withdrawing the azeotrope containing the water from the distillationzone,

continuing the presence of the pancreas in the body of organic liquidfat solvent until substantially all the fat of the fresh pancreas isdissolved in said solvent, and

separating the substantially dried and substantially defatted pancreatictissue material from the body of solvent to provide a stable insulincontaining material. 6. A method as in claim Sin which the freshpancreas is contacted with an acid selected from the group consisting ofsulfuric, hydrochloric and nitric acids,

the acid contacted pancreas is ground to provide thorough mixing of thematerial and the acid,

delivering to the enzyme deactivated pancreas mixture several times itsown volume of boiling ethylene dichloride solvent so that sufficientsolvent is present to both remove the water from the pancreas as anazeotrope and to dissolve the fat of the pancreas.

7. A method as in claim 5 in which the pancreatic material is selectedfrom the group consisting of beef and pork pancreas, and the fat organicsolvent is ethylene dichloride,

said pancreatic material is separated from the fat organic solvent bydraining the solvent away from the distillation zone,

the separated pancreatic material is treated with a plurality ofethylene dichloride washing steps, and

the separated pancreatic material is dried to remove ethylenedichloride.

8. A method of obtaining insulin in which substantially dry andsubstantially defatted pancreatic material obtained by the process ofclaim 5 is utilized as a starting material for the production of insulinby process steps which include dispersing the said pancreatic materialin an aqueous medium for a time sufficient to extract substantially allthe insulin therein,

separating the substantially extracted tissue material from the aqueousmedium, and precipitating the extracted insulin in said aqueous medium.

@223 v Q UNITED STATES PATENT OFFICE v CERTIFICATE OF CQRRECTION PatentNo. 31676552 Dated July ll, 1972 Inventor-(s) r Levln It iscertifiedthat error enpeers in the above-identified patent and that said Letters.Patent are hereby corrected as shown below:

- e t Ezra Levin, Champaign, Illinois 9 Vio Bin Corporation, Chainpaign,Illinois Signed and sevaled this 15th day of May 1977 Attest:

EDWARD M.=.FLETCHER,JR. ROBERT. GOTISCHALK Attesting Officer tCommissioner of Patents

2. A product as in claim 1 in which the dry product has a water contentof less than 6 percent by weight, and has a fat content of less than 3percent by weight.
 3. A product as in claim 2 in which the comminutedtissue material is in the form of a powder.
 4. A product as in claim 1in which the pancreatic material is selected from the group consistingof beef and pork pancreas.
 5. A method for preparing a stable insulinsource material from which active insulin may be later separated withoutany substantial changes in the insulin levels present in an originalsource of fresh pancreas, including the steps of contacting a body offresh pancreas containing insulin, pancreatic enzymes, water and fatwith acid of a type and in sufficient amount to deactivate substantiallyall the pancreatic enzymes, combining the enzyme deactivated freshpancreas in the comminuted state with a body of boiling organic liquidfat solvent selected from the group consisting of ethylene dichloride,trichloroethylene, propylene dichloride, perchloroethylene, benzene,hexane, pentane, toluene, cyclopentane and cyclohexane in a distIllationzone which is depressurized so that the said organic liquid fat solventboils below 100* F, said organic liquid fat solvent forming an azeotropewith water, withdrawing the azeotrope containing the water from thedistillation zone, continuing the presence of the pancreas in the bodyof organic liquid fat solvent until substantially all the fat of thefresh pancreas is dissolved in said solvent, and separating thesubstantially dried and substantially defatted pancreatic tissuematerial from the body of solvent to provide a stable insulin containingmaterial.
 6. A method as in claim 5 in which the fresh pancreas iscontacted with an acid selected from the group consisting of sulfuric,hydrochloric and nitric acids, the acid contacted pancreas is ground toprovide thorough mixing of the material and the acid, delivering to theenzyme deactivated pancreas mixture several times its own volume ofboiling ethylene dichloride solvent so that sufficient solvent ispresent to both remove the water from the pancreas as an azeotrope andto dissolve the fat of the pancreas.
 7. A method as in claim 5 in whichthe pancreatic material is selected from the group consisting of beefand pork pancreas, and the fat organic solvent is ethylene dichloride,said pancreatic material is separated from the fat organic solvent bydraining the solvent away from the distillation zone, the separatedpancreatic material is treated with a plurality of ethylene dichloridewashing steps, and the separated pancreatic material is dried to removeethylene dichloride.
 8. A method of obtaining insulin in whichsubstantially dry and substantially defatted pancreatic materialobtained by the process of claim 5 is utilized as a starting materialfor the production of insulin by process steps which include dispersingthe said pancreatic material in an aqueous medium for a time sufficientto extract substantially all the insulin therein, separating thesubstantially extracted tissue material from the aqueous medium, andprecipitating the extracted insulin in said aqueous medium.